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Studies of the conformational stability of invasion plasmid antigen B from Shigella.

ABSTRACT: Shigella spp. are the causative agent of shigellosis, the second leading cause of diarrhea in children of ages 2-5. Despite many years of research, a protective vaccine has been elusive. We recently demonstrated that invasion plasmid antigens B and D (IpaB and IpaD) provide protection against S. flexneri and S. sonnei. These proteins, however, have very different properties which must be recognized and then managed during vaccine formulation. Herein, we employ spectroscopy to assess the stability of IpaB as well as IpgC (invasion protein gene), IpaB's cognate chaperone, and the IpaB/IpgC complex. The resulting data are mathematically summarized into a visual map illustrating the stability of the proteins and their complex as a function of pH and temperature. The IpaB/IpgC complex exhibits thermal stability at higher pH values but, though initially stable, quickly unfolds with increasing temperature when maintained at lower pH. In contrast, IpaB is a much more complex protein exhibiting increased stability at higher pH, but shows initial instability at lower pH values with pH 5 showing a distinct transition. IpgC precipitates at and below pH 5 and is stable above pH 7. Most strikingly, it is clear that complex formation results in stabilization of the two components. This work serves as a basis for the further development of IpaB as a vaccine candidate as well as extends our understanding of the structural stability of the Shigella type III secretion system.

SUBMITTER: Choudhari SP

PROVIDER: S-EPMC3649268 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Studies of the conformational stability of invasion plasmid antigen B from Shigella.

Choudhari Shyamal P SP Kramer Ryan R Barta Michael L ML Greenwood Jamie C JC Geisbrecht Brian V BV Joshi Sangeeta B SB Picking William D WD Middaugh C Russell CR Picking Wendy L WL

Protein science : a publication of the Protein Society 20130326 5

Shigella spp. are the causative agent of shigellosis, the second leading cause of diarrhea in children of ages 2-5. Despite many years of research, a protective vaccine has been elusive. We recently demonstrated that invasion plasmid antigens B and D (IpaB and IpaD) provide protection against S. flexneri and S. sonnei. These proteins, however, have very different properties which must be recognized and then managed during vaccine formulation. Herein, we employ spectroscopy to assess the stabilit ...[more]

PMID: 23494968

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Project description:An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.

Project description:Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. KEY POINTS : • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli-produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice.

Dataset Information

Studies of the conformational stability of invasion plasmid antigen B from Shigella.

Publications

Studies of the conformational stability of invasion plasmid antigen B from Shigella.

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